Bearded capuchin monkeys as a model for Alzheimer’s disease

The absence of a natural animal model is one of the main challenges in Alzheimer’s disease research. Despite the challenges of using nonhuman primates in studies, these animals can bridge mouse models and humans, as nonhuman primates are phylogenetically closer to humans and can spontaneously develop AD-type pathology. The capuchin monkey, a New World primate, has recently attracted attention due to its skill in creating and using instruments. We analyzed one capuchin brain using structural 7 T MRI and performed a neuropathological evaluation of three animals. Alzheimer-type pathology was found in the two of the capuchins. Widespread β-amyloid pathology was observed, mainly in focal deposits with variable morphology and a high density of mature plaques. Notably, plaque-associated dystrophic neurites associated with disruption of axonal transport and early cytoskeletal alteration were frequently found. Unlike in other species of New World monkeys, cerebral arterial angiopathy was not the predominant form of β-amyloid pathology. Additionally, abnormal aggregates of hyperphosphorylated tau, resembling neurofibrillary pathology, were observed in the temporal and frontal cortex. Astrocyte hypertrophy surrounding plaques was found, suggesting a neuroinflammatory response. These findings indicate that aged capuchin monkeys can spontaneously develop Alzheimer-type pathology, indicating that they may be an advantageous animal model for research in Alzheimer’s disease.

β-Amyloid deposits AD-type pathology was observed in 29-and 33-year-old but not in 9-year-old capuchin.In both aged capuchin monkeys, β-amyloid (βA) deposits were observed in the hippocampus, amygdala, basal ganglia, and all cortical areas except the occipital cortex, where only amyloid angiopathy was found.A moderate density of diffuse and focal immunoreactive deposits was found.Although βA deposits were observed in both hemispheres, a greater burden was noted in the left hemisphere than in the right hemisphere in monkey S2.
Widespread βA immunoreactivity was observed in the form of diffuse and focal deposits (Fig. 3).Most were focal, with a predominance of mature rather than primitive plaques (Fig. 3).Nevertheless, variable plaque morphology was observed, predominantly in the form of classical dense-cored plaques with a core-space-corona, coarse-grained plaque, burnt-out plaque, and juxtavascular plaque (Fig. 3).Plaques with amyloid cores were also observed via routine staining (Fig. 3).
In addition, in many cortical areas, βA deposits were found in the form of amyloid angiopathy in leptomeningeal and cortical vessels with involvement of not only arterioles but also capillaries (Fig. 3).

Other neuropathological alterations
In addition to the presence of βA and Ptau aggregates, small intranuclear inclusions (Marinesco bodies) were observed in neurons of the substantia nigra (Fig. 4).These eosinophilic spherical nuclear inclusions were strongly immunoreactive for p62 (Fig. 4).Additionally, a yellowish-brown Periodic acid-Schiff (PAS)-positive granular pigment, consistent with lipofuscin, was observed in the neuronal and glial cells via hematoxylin-eosin staining (Fig. 4).This PAS-positive pigment was positive for β-amyloid and p62.
Immunohistochemistry using TDP-43 and phosphorylated α-synuclein antibodies failed to reveal immunoreactive aggregates of these proteins in cortical, limbic, or brainstem regions.

Discussion
AD-type pathology was found in two aged Sapajus libidinosus monkeys.Diffuse and focal βA deposits were observed in cortical and limbic areas.Notably, the deposits were usually more focal than diffuse.The density of mature plaques was greater than that of primitive plaques.These findings contrast with those of previous studies describing the predominance of diffuse deposits and primitive plaques in other New World monkeys, such as squirrel monkeys and marmosets 7,9,32 .
In addition, in our cases, neuritic plaques were frequently found in not only cortical but also subcortical areas such as the hippocampus, amygdala, and basal ganglia, contrasting with the predominance of diffuse deposits and primitive plaques described in squirrel monkeys and marmosets.Additionally, it is important to highlight the high density of mature plaques, even when compared to the density of cerebral amyloid angiopathy, and the presence of different morphologies similar to those observed in human brains in AD.Interestingly, cerebral amyloid angiopathy was not the predominant form of βA pathology in these two capuchin monkeys, unlike that observed in other New World species 7,9 .
In addition, abnormal aggregates of Ptau were observed in hippocampus, temporal and frontal cortex, resembling neurofibrillary pathology.The Ptau immunoreactive pathology is very similar to that observed in the brains of human AD patients 33 .
The lack of Ptau in most dystrophic neurites surrounding classical neuritic plaques and the sparse density of neurofibrillary pathology can indicate the early stages of the disease 34 .However, these findings can be associated with species differences in the tau protein 6,35 .
Despite the low burden of Ptau pathology, the finding that these species can spontaneously develop AD-type pathology similar to that observed in humans is highly relevant.Additionally, the observed microglial activation and astrocyte hypertrophy indicate a neuroinflammatory response to AD-type pathology that can contribute to neurodegeneration or play a protective role 36 .Our findings identified a nonhuman primate species that may represent a new lower primate model appropriate for AD studies.
Additionally, studies of Marinesco bodies and lipofuscin accumulation have shown that they are aging-related microscopic findings, but their relationship with neurodegeneration is still unclear 37,38 .However, they are markers of cellular dysfunction and are associated with the main mechanisms for degrading proteins implicated in neurodegeneration, highlighting the value of the capuchin monkey as a natural model 37,38 .
The three animals participated in cognitive tests carried out at different times with satisfactory performance.However, in the present study, we obtained only scant information about cognitive decline or behavioral changes www.nature.com/scientificreports/associated with aging in the two older capuchin monkeys.In future studies, we aim to obtain additional data to associate cognition and behavior with AD markers.
A literature review revealed that Sapajus sp. is a primate with many qualities that recommend its inclusion among AD animal models, with some advantages relative to others.Based on its genetic, behavioral, and morphological characteristics, the capuchin monkey is phylogenetically more closely related to humans than are other New World primates 19,39 .
Compared to Old World primates, capuchins are relatively small, as well as simpler and less expensive to maintain in captivity.Sapajus libidinosus surprised researchers with its skill in creating and using instruments 22,23 .It is a very curious and motivated animal that can be evaluated using neuropsychological tests similar to those used in other primates and humans 16,[26][27][28] .
Compared with those of other primates, such as Saimiri 41 and marmosets 2 , which have been proposed for use as animal models of AD, Sapajus has much more substantial cerebral cortex development and remarkable cognitive ability 14,16,[21][22][23] .Moreover, the tau isoform expression pattern in marmosets may be more like that in mice than in humans 38 .
Using monkeys as natural models of AD is challenging due to the long time required for these animals to reach old age 2 .However, identifying cognitive decline and plasma biomarkers of βA and Ptau pathology in these animals may be highly relevant for developing evolutive biomarkers and new treatments for AD.It should also be considered that the Cebidae family, of which the bearded capuchin monkey is a member, is one of the less threatened primate families 40 .To conclude, Sapajus libidinosus should be considered an advantageous animal model for AD research.

Materials and methods
This study has been approved by the local ethics committees.All animal care and experimental protocols were approved by the Ethics Committee for Animal Use of the University of Brasilia (SEI 23106.123230/2023-68).We did not perform any type of experiment relevant to this study during the lives of these primates.Management and maintenance to support the good welfare of these animals in captivity were carried out according to the Brazilian regulations for the Care and Use of Animals for Scientific Purposes established by the National Council for Control Animal Experimentation (CONCEA) (Lei Arouca 11.794/2008).The housing and maintenance conditions of the primates followed the laws and regulations of the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA).Additionally, protocols and methods performed in the University of São Paulo were in accordance with Brazilian and international guidelines.
In 2016, a cooperative study was launched between the Department of Neurology at the University of São Paulo (USP) and the Primate Center at the University of Brasília (UnB) to investigate the presence of AD pathological markers in the brains of capuchin monkeys.
The Primate Center of the UnB aims to provide a captive breeding colony of Brazilian neotropical primates for ethological and biomedical research.This center is located on a farm of 4.340 ha (16030′′ S, 46030′′ W) in a protected area of an ecological reserve and houses primates in cages surrounded by nearby semideciduous tropical native gallery forests under natural light, temperature, and moisture conditions.
Upon arrival at the Primate Center, all monkeys undergo a thorough health assessment to verify their disease-free status, physical well-being, and readiness for integration.This entails a quarantine period, physical examination, weight and vital signs assessment, physical check-up, blood tests, fecal examination, and behavioral observation/socialization assessment.If anomalies are observed during a physical exam, supplementary tests such as X-ray or ultrasound may be performed.Postintegration, ongoing monitoring through regular health checks ensures the continual well-being of all individuals under the Primate Center's care.
The brains of three adult Sapajus libidinosus capuchin monkeys (2 males and 1 female) kept at the Primatology Center and who died of natural causes (the nine-year-old monkey died of pneumonia) were used for this study.The Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA) collected the animals and sent them to the Primatology Center, where they arrived in adulthood.Therefore, the ages of the animals were estimated based on their morphological characteristics at the time of arrival plus the length of stay at the Primatology Center.The monkeys were kept in pairs in cages (4 m long × 2.9 m wide × 2 m high) with two concrete walls separating adjacent cages and wire mesh as the front, back, and roof, forming an external/semiinternal housing system.Each cage contained a suspended wooden nest box, several wooden perches at different heights, a food tray where the animals were fed, and a thick layer of natural dry leaves on the floor.The animals had olfactory and acoustic contact but not visual contact with other colony members.Fresh fruits and vegetables were provided daily from 7:30 a.m. until 5:00 p.m. Primate food and fresh water were provided ad libitum.The animals received ongoing veterinary monitoring and underwent clinical evaluations and monthly weighings.
A complete neuropathological evaluation was performed for the three animals in the study (9-and 33-year-old males and a 29-year-old female).Moreover, a structural 7 T magnetic resonance image (7 T MRI) was acquired from only one patient (a 29-year-old female) before and after brain slicing (Table 1).
An experienced veterinarian performed the brain extractions from the monkeys after their deaths.Brain tissue was placed in 4% buffered paraformaldehyde within 12 h of death and fixed for three weeks.Consecutive coronal sections from the fixed brain were embedded in paraffin, and 5 μm sections from paraffin blocks were used for staining and immunohistochemistry evaluation.All brain sections were stained with hematoxylin and eosin.

Study design
The present study is a prospective descriptive study.Our aim was to investigate the presence of neuropathological changes characteristic of Alzheimer's disease in capuchin monkeys who died of natural causes.We did not perform any type of experiment relevant to this study during the lives of these primates.

Sample size
Three adult Sapajus libidinosus capuchin monkeys (2 males and 1 female) kept at the Primatology Center and who died of natural causes were included in this study.The Primatology Center of the University of Brasilia is located on a farm of 4.340 ha (16030" S, 46030" W) in a protected area of an ecological reserve and houses primates in cages surrounded by nearby semideciduous tropical native gallery forests under natural light, temperature, and moisture conditions.The main objective of the center is to provide a captive breeding colony of Brazilian neotropical primates for ethological and biomedical research.Animals arrive at the Primatology Center in adulthood.Therefore, the ages of the animals were estimated based on their morphological characteristics at the time of arrival, plus the length of their stay at the Primatology Center.The animals were kept in pairs in cages (4 m long × 2.9 m wide × 2 m high), which consisted of two concrete walls separating adjacent cages and wire mesh at the front, back, and roof, forming an external/ semi-internal housing system.Fresh fruits and vegetables were provided daily from 7:30 a.m. until 5:00 p.m.The animals were subjected to ongoing veterinary monitoring and clinical evaluation and monthly weighing.

Inclusion criteria
Sapajus libidinosus at the Primatology Center of the University of Brasilia who died from natural causes.

Exclusion criteria
Animals not sampled within 24 h postmortem.

Randomization
Not applicable.

Not applicable.
Outcome measures Not applicable.

Experimental animals Not applicable.
Experimental procedures Not applicable.(Neuroimaging and neuropathological studies were carried out after death of natural causes.)

Results
We described the presence of Alzheimer's type pathology in the two aged capuchin monkeys (29-and 33-yearold monkeys).

Figure 1 .
Figure 1.Macroscopic images.(a) Superior, (b) inferior, and (c) lateral views of the three capuchin monkey brains showing cortical gyrification.(d) Coronal section of the hippocampus at the level of the lateral geniculate nucleus.Scale unit: centimeters.

Figure 2 .
Figure 2. 7 T ex situ brain magnetic resonance image (MRI) of a 29-year-old capuchin monkey.(a) Axial brain 7 T MRI image showing cortical gyrification.(b) In a T2-weighted image obtained from a 7 T MRI of a coronal slice, cortical lamination of the frontal cortex (blue arrow) and connections between the caudate and putamen (orange arrow) were observed.The claustrum can also be well identified (green arrow).(c) A macroscopic coronal slice at the same level as in (b).

Table 1 .
Monkeys characteristics.a Estimated age.b Postmortem.